Antimicrobial Activity of Diffusible and Volatile Metabolites Emitted by Beauveria bassiana: Chemical Profile of Volatile Organic Compounds (VOCs) Using SPME-GC/MS Analysis

The genus Beauveria includes important entomopathogenic and endophytic fungi; among them, Beauveria bassiana is the most studied species. However, there is little knowledge regarding their antimicrobial activity. The current research has been conducted to evaluate the in vitro antagonistic activity of B. bassiana and the antimicrobial efficacy of its Exo and Endo metabolites against Bacillus cereus, B. megaterium, Clavibacter michiganensis (Gram positive bacteria, G+ve), Xanthomonas campestris, Pseudomonas aeruginosa and P. fluorescence (Gram negative bacteria, G−ve). In addition, solid-phase microextraction (SPME) was coupled with Gas Chromatography-Mass Spectrometry (GC/MS) to qualitatively measure the volatile organic compounds’ (VOCs) metabolic profile of the most efficient studied isolate of B. bassiana. The obtained results showed that the isolate UniB2439-3 has a promising antibacterial effect against most of the studied target bacteria. An SPME-GC/MS analysis of VOCs revealed the presence of ethanol, butanal,2-methyl, 2,4-dimethyl-1-heptene, octane, 4-methyl and β-elemene as the dominant bioactive compounds. The results demonstrated that the efficient isolate of B. bassiana can be potentially used as a biocontrol agent against several bacteria, especially G+ve ones.


Introduction
Genus Beauveria includes entomopathogenic and endophytic fungi, which are widespread in different habitats [1][2][3]. Furthermore, many researchers have reported that fungi in the genus Beauveria can produce enzymes for biotransformation and biodestructors [4][5][6]. Some species of this genus, such as B. bassiana and B. brongniartii, are able to produce mycoinsecticides [7]. B. bassiana is also a beneficial microorganism (BM) and endophytic fungus (EF) in several crops and is commonly known as biological control agent against a variety of agricultural pests [3,[8][9][10]. The application of B. bassiana has many advantages, such as being a form of eco-friendly management compared to chemical pesticides, and being harmless to human health [8,[10][11][12]. For decades, several scientists have reported the importance of B. bassiana in reducing a range of nuisance insects, where it can induce direct insect mortality [2,13,14] and reach a 90% reduction in life-time fecundity [15].
A recent study, conducted by Barra-Bucarei et al. [16] to evaluate the colonization ability of native endophytes of different strains of B. bassiana and their antifungal effect against Botrytis cinerea in tomato and chili pepper, concluded that all studied strains had significant in vitro antagonism against B. cinerea. The same study reported that the native The PCR amplification for β-tubulin genes with Bt2a/Bt2b produced, for each gDNA extracted from the above five isolates (UniB2439-1, UniB2439-2, UniB2439-3, UniB2439-4, and UniB2439-5), amplicons with a nucleotide length of about 330 bp. The DNA extracted from the same five isolates and amplified with ITS5/ITS4 for rRNA produced amplicons with a nucleotide length of about 600 bp. No amplification was observed in the case of the negative control. The amplicons were directly sequenced (BMR Genomics, Padova, Italy), and the obtained sequences were compared with those available in the GenBank nucleotide archive, showing a high similarity percentage (97.29%) with the sequences AB829899, AB829898, and CP045886.1, and those available for B. bassiana in the NCBI database using Basic Local Alignment Search Tool software (BLAST) (Bethesda, Rockville Pike, MD, USA) [27]. The five obtained sequences were deposited in the NCBI GeneBank with accession numbers FR989662-FR989666. The phylogenetic analysis confirmed the identification of the five studied isolates as B. bassiana ( Figures S1 and S2).

Antagonistic Activity of B. bassiana Isolates
The preliminary results showed that all tested isolates of B. bassiana had antagonistic effects against most tested bacterial strains, as illustrated in Table S1. UniB2439-3 was the most efficient isolate. In fact, this isolate showed the most significant effect against Bacillus cereus and Clavibacter michiganensis, a moderate effect against B. megaterium and a low effect against Xanthomonas campestris and Pseudomonas fluorescens. This isolate did not show any activity against P. aeruginosa ( Figure 1). Therefore, the UniB2439-3 isolate was selected for further biological and chromatographic analyses.

Molecular Identification of the Studied Isolates of Beauveria
The PCR amplification for β-tubulin genes with Bt2a/Bt2b produced, for each gDNA extracted from the above five isolates (UniB2439-1, UniB2439-2, UniB2439-3, UniB2439-4, and UniB2439-5), amplicons with a nucleotide length of about 330 bp. The DNA extracted from the same five isolates and amplified with ITS5/ITS4 for rRNA produced amplicons with a nucleotide length of about 600 bp. No amplification was observed in the case of the negative control. The amplicons were directly sequenced (BMR Genomics, Padova, Italy), and the obtained sequences were compared with those available in the GenBank nucleotide archive, showing a high similarity percentage (97.29%) with the sequences AB829899, AB829898, and CP045886.1, and those available for B. bassiana in the NCBI database using Basic Local Alignment Search Tool software (BLAST) (Bethesda, Rockville Pike, MD, USA) [27]. The five obtained sequences were deposited in the NCBI GeneBank with accession numbers FR989662-FR989666. The phylogenetic analysis confirmed the identification of the five studied isolates as B. bassiana ( Figures S1 and S2).

Antagonistic Activity of B. bassiana Isolates
The preliminary results showed that all tested isolates of B. bassiana had antagonistic effects against most tested bacterial strains, as illustrated in Table S1. UniB2439-3 was the most efficient isolate. In fact, this isolate showed the most significant effect against Bacillus cereus and Clavibacter michiganensis, a moderate effect against B. megaterium and a low effect against Xanthomonas campestris and Pseudomonas fluorescens. This isolate did not show any activity against P. aeruginosa ( Figure 1). Therefore, the UniB2439-3 isolate was selected for further biological and chromatographic analyses.

Antimicrobial Activity of Exo-and Endo-Diffusible Metabolites
The antimicrobial activity of extracted metabolites was determined following the disc-diffusion method. The obtained results for the metabolites extracted from the selected isolate of B. bassiana UniB2439-3 showed that extracellular metabolites (Exo-ME) were more able to inhibit the growth of most tested bacterial strains than the endocellular (Endo-ME) ( Table 1). In particular, Exo-ME showed the most significant activity against C. michiganensis. In addition, both extracts showed equal activity against X. campestris, whereas only Endo-ME showed antibacterial activity against P. aeruginosa. On the other hand, Endo-ME was not active against B. cereus or C. michiganensis.

Antimicrobial Activity of Exo-and Endo-Diffusible Metabolites
The antimicrobial activity of extracted metabolites was determined following the disc-diffusion method. The obtained results for the metabolites extracted from the selected isolate of B. bassiana UniB2439-3 showed that extracellular metabolites (Exo-ME) were more able to inhibit the growth of most tested bacterial strains than the endocellular (Endo-ME) ( Table 1). In particular, Exo-ME showed the most significant activity against C. michiganensis. In addition, both extracts showed equal activity against X. campestris, whereas only Endo-ME showed antibacterial activity against P. aeruginosa. On the other hand, Endo-ME was not active against B. cereus or C. michiganensis. G−ve X. campestris 9.5 ± 2.5 ab 9.0 ± 1.9 a 23.5 ± 1.7 ab P. aeruginosa 0.0 ± 0.0 c 6.5 ± 2.8 ab 10.6 ± 0.7 c P. fluorescens 6.5 ± 1.5 b 4.5 ± 1.7 b 12.3 ± 0.9 c Values followed by different letters in each column for each tested extract against all tested bacteria are significantly different at p < 0.05. Data are expressed as the mean of the inhibition zone diameter (mm) for three replicates ± SDs compared to controls ± SDs.

Antibacterial Activity of Volatiles Metabolites
The in vitro antibacterial activity of the volatile metabolites eventually emitted by B. bassiana was evaluated against both the grown-visible colonies (GVC) and aqueous suspension (AQS) of each tested bacterial strain. The results of an in vitro bioactivity assay demonstrated that the studied isolate of B. bassiana (UniB2439-3) produced bioactive volatile metabolites that were able to significantly reduce the growth of tested bacterial strains compared to tetracycline (positive control). In particular, the efficacy of the produced volatile substances was high against the AQS of all tested bacterial strains, higher than GVC ( Table 2). In addition, the highest antibacterial activity was observed in the case of GVC against B. megaterium (G+ve) and P. fluorescens (G−ve), estimated at 77.5 and 52.5%, respectively. On the other hand, the highest antibacterial activity in the case of AQS was observed against B. megaterium (G+ve) and P. aeruginosa (G−ve), estimated at 92.0 and 87.5%, respectively.

Discussion
Research has been conducted recently to overcome the multi-drug-resistant (MDR) microorganisms to different antibiotics and chemotherapeutic agents [29]. Hence, the search for new active and natural agents has attracted great interest, particularly for human health and environmental protection [30]. Beauveria, one of the most studied genera among entomopathogenic fungi, has various biological applications as a growth-promoting agent or insecticide [9,10,31,32].
The results revealed that both extracts are less effective than the control (tetracycline); nevertheless, they can be regarded as hopeful and prospective antimicrobial agents or as alternatives for synthetic pesticides. On the other hand, considering the higher activity of Exo-ME against C. michiganensis and the equal activity of both extracts against X. campestris, it would be beneficial to consider the potential synergistic effects of combining the two extracts in future studies.
On the other hand, Barra-Bucarei et al. [16] studied the antifungal activity of 10 native strains of B. bassiana, an endophyte for tomato and chili pepper, and observed that the majority of the studied native strains were able to colonize tomato and chili pepper tissues and showed a promising antagonistic effect against B. cinerea.
The capacity of B. bassiana to produce several volatile metabolites with possible antimicrobial effects is in agreement with previous bibliographic research investigating its antagonistic effect against several phytopathogens [9,17,33]. In fact, the bibliographic The eventual fragmentation of the acquired volatile metabolites, as described here, is also shown in Table 3. Beauvericin was fragmented into diethyl phthalate with 90%, the major dominant constituent, carbon dioxide, and nitrous oxide. Bassianolide was converted to butanal, 3-methyl-with 81% or 1-butanol, 3-methyl-with 83%. Regarding bassianin, GC-MS analysis showed that this compound was converted into 2,4-Dimethyl-1-heptene with 90%. Beauveriolide was fragmented into butanal, 3-methyl with 81%, carbon dioxide and nitrous oxide. Regarding cyclosporine, the results demonstrated that this compound was fragmented into butanal, 2-methyl with 90%, butanal, 3-methyl with 81%, 1-butanol, 3-methyl with 83%, carbon dioxide and nitrous oxide.

Discussion
Research has been conducted recently to overcome the multi-drug-resistant (MDR) microorganisms to different antibiotics and chemotherapeutic agents [29]. Hence, the search for new active and natural agents has attracted great interest, particularly for human health and environmental protection [30]. Beauveria, one of the most studied genera among entomopathogenic fungi, has various biological applications as a growth-promoting agent or insecticide [9,10,31,32].
The results revealed that both extracts are less effective than the control (tetracycline); nevertheless, they can be regarded as hopeful and prospective antimicrobial agents or as alternatives for synthetic pesticides. On the other hand, considering the higher activity of Exo-ME against C. michiganensis and the equal activity of both extracts against X. campestris, it would be beneficial to consider the potential synergistic effects of combining the two extracts in future studies.
On the other hand, Barra-Bucarei et al. [16] studied the antifungal activity of 10 native strains of B. bassiana, an endophyte for tomato and chili pepper, and observed that the majority of the studied native strains were able to colonize tomato and chili pepper tissues and showed a promising antagonistic effect against B. cinerea.
The capacity of B. bassiana to produce several volatile metabolites with possible antimicrobial effects is in agreement with previous bibliographic research investigating its antagonistic effect against several phytopathogens [9,17,33]. In fact, the bibliographic research revealed that the genus Beauveria produced some interesting metabolites, such as oosporein, beauvericin, bassianolide, bassianin, beauveriolide, bassiacridin and cyclosporine, with notable insecticide and antimicrobial actions [34][35][36][37][38][39][40]. Among the different bioactive metabolites produced by B. bassiana, several studies revealed that beauvericin and oosporein evidenced remarkable antibiotic and antifungal properties [39,40], which are probably involved in the microbial growth-inhibition observed in the bioassay presented in this study. Furthermore, Wang and Xu [41] reported that beauvericin was one of the active constituents of B. bassiana and confirmed to have antimicrobial activity and anti-tumor effects, especially against human leukemia. In another study, conducted by Manning and Wyatt [42], the results demonstrated that oosporein, extracted from the broth cultures of Beauveria and Chaetomium, has been identified as a toxic substance for plants and poultry.
The possible mechanism of volatile antimicrobial effects, in general, may be explained by the potential of volatiles to flow across a structure of soil gaps since they are active in both gaseous and liquid phases and have the potential of revolatization after flowing through water-saturated pores [26]. However, because of their high vapor pressure, volatiles mostly traverse through vapor diffusion. However, this process is regulated by the intrinsic chemical characteristics of each VOC and also the physicochemical characteristics of adjacent soil, which affect adsorption, desorption, and degradation.
In particular, 2,4-dimethyl-1-heptene showed antimicrobial activity, as reported by Mannaa and Kim [45]. In addition, 2,3,3-trimethyl-Octane, which is close to octane, 4methyl, showed a higher docking energy than the commercial anti-inflammatory drug, as reported by Saravanakumar et al. [46]. Methyl-1-butanol was identified as one of the primary volatile chemicals released from active cultures of Enterobacter agglomerans [47]. Salih et al. [48] also reported that butanol, among the major constituents detected in Coccoloba peltate, showed notable antioxidant and cytotoxic effects.
Generally, the mechanism of the antimicrobial activity of several terpenes is highly related to their lipophilic properties, which enable them to dissolve in the phospholipid layers of a microbial cell membrane [53]. Particularly, natural sesquiterpenes such as βelemene, which originate from plants and microorganisms, showed promising antimicrobial activity [54,55]. A recent study conducted by Monga and Sharma [56] reported that βelemene and R-limonene play an essential role in degrading the microbial cell wall, altering the expressions of dprE1 and clgR genes, which are responsible for cell wall synthesis and cell membrane preservation, respectively.
Some recent studies reported on the promising cytotoxic effects of β-elemene, which can inhibit cell proliferation, arrest cell cycle, and induce cell apoptosis or autophagy [57]. B-elemene is one of the most promising inhibitors of the glycolysis rate-limiting enzyme, especially (PKM2), through its interference with tumor glycolysis, which is considered one of the most important recent strategies for treating tumors [58,59]. In fact, research has reported that inhibition of tumor growth and proliferation can be achieved by downregulating the expression of the PKM2 enzyme [60]. In addition, Pan et al. [61] pointed to the role of β-elemene in inhibiting breast cancer cell migration by converting dimer and tetramer forms of PKM2, inhibiting aerobic glycolysis, and reducing the utilization of glucose and the production of lactic acid for tumor cell growth.

Antagonistic Activity
The antagonistic activity of the five studied isolates of B. bassiana was evaluated against some pathogenic bacteria. All tested isolates were obtained from the pure cultures conserved in the collection of SAFE and identified using morphological and molecular methods. The tested bacteria strains are listed in Table 4. An antibacterial assay was carried out as described by Elshafie et al. [65]. A fungal disc of approximately 0.5 cm from the fresh PDA culture (96 h) of each studied isolate of B. bassiana was deposited in the center of the King B nutrient media (KB) Petri dish and incubated for 16 h at 22 ± 2 • C. Successively, a suspension of soft-agar (0.7%) of each tested bacteria at 10 8 CFU/mL was sprayed over the plates using Eco-Spray Ecological Aerosol (Seidden Identification, Madrid, Spain). All plates were incubated at 30 • C for 24 h. Two KB plates inoculated only with each tested bacteria were used as a negative control. The experiment was run in triplicate, and the diameter of the inhibition zone was measured with a caliber and recorded as the mean ± SD (n = 3). The antagonistic bacterial activity percentage (ABP%) was calculated using Equation (1): where ABP is the antagonistic bacterial activity; D.iz: is the diameter of inhibition zones in cm; D.ct: is the diameter of control plates in cm.

Extraction of Secondary Metabolites
On the basis of the preliminary antagonistic assay, the most efficient isolate (UniB2439-3) of B. bassiana was selected for successive studies. For this purpose, 2 mL of the fungal suspension (10 6 spores/mL) of the above isolate was used to inoculate 500 mL SDY broth nutrient media and then incubated for 7 days at 25 • C in agitation (180 rpm). Both Exo-ME and Endo-ME were extracted from the broth culture after the incubation period.
For Endo-ME, the incubated broth culture was centrifuged at 40,000× g for 15 min, and the pellet (2 g) was collected, resuspended in 50 mL of Limonene (CAS 138-86-3-Aldrich, Steinheim, Germany) and shaken for 2 h; after this, the solvent was evaporated using the rotary-evaporator (Heidolph WB2000, Schwabach, Germany). The residue was resuspended in 2 mL of sterile distilled water (SDW), following the Solid Phase Extraction (SPE) method using a C-18 column (Thermo Scientific, Rockwood, TN, USA), and recovered using 1 mL methanol to reach the final original concentration of (20 mg/mL) [24].
For Exo-ME, the supernatant (250 mL) obtained from the above centrifugation step was filtered using 0.22 µm (syringe filter-hydrophilic, Minisart, Goettingen, Germany) and extracted using a separator funnel containing 250 mL ethyl acetate/ethanol (70:30; v/v) and shacked for 15 min. The organic phase was filtered through a filter paper (Whatman, Ø. 25 mm, Merck KGaA, Darmstadt, Germany) and evaporated using the rotary-evaporator. The dry residue (50 mg) was resuspended in 2 mL SDW, extracted through SPE using a C-18 column, and recovered using 1 mL methanol to reach the final original concentration of (16 mg/mL) [24].

Antibacterial Activity of Diffusible Metabolites
The antibacterial activity of both metabolite extracts, compared with the same bacteria strains used for the initial antagonistic assay, is listed in Table 4.
Disc diffusion assay. An antibacterial test of both metabolite extracts produced by the most bioactive isolate UniB2439-3 was carried out following the disc-diffusion method, as described by Elshafie et al. [66] and Sofo et al. [67]. A bacterial suspension of each tested bacteria was prepared in sterile distilled water adjusted at 10 6 CFU/mL (OD ≈ 0.2 nm) using UV-Spectrophotometer (Amersham, Ultraspec 1100 pro/500 pro, UK). A total of 4 mL of bacterial suspension mixed with soft agar 0.7% (9:1; v/v) was poured over each KB plate (Ø 9 cm). Blank discs of 6 mm (OXOID, Milan, Italy) were then placed over the plates and 15 µL from each tested metabolite extract (Exo-ME 16 mg/mL and Endo-ME 20 mg/mL) were carefully applied to the discs. Tetracycline (1600 µg/mL) was used as a positive control. The experiment was performed in triplicate, and the antibacterial activity was estimated by measuring the diameter of the inhibition zone in mm ± SDs compared to the positive control.

Antibacterial Activity of Volatiles Metabolites
The tested bacterial strains were initially subcultured on 14 mL KB medium in Petri dishes and incubated at 37 • C for 24 h. The most efficient isolate of B. bassiana (UniB2439-3) was cultured on PDA media (14 mL Petri dishes) and incubated at 22 • C for 96 h. The test was performed according to Wan et al. [68] using a double-dish chamber containing the studied strain of B. bassiana in one downward dish of KB (Ø 90 mm), and the tested bacterial strains were singularly inoculated on the upward dish, either by direct colony inoculation or the spread of 50 µL of an aqueous suspension (10 7 CFU/mL). In brief, the direct inoculation of colonies was carried out using a sterile swab to homogenize the colonies over the KB surface. The concentration of the aqueous bacterial suspension was adjusted using turbidimetry. The chamber was sealed with Parafilm™ and incubated at 37 • C in darkness for 48 h. The antibacterial activity of the eventually produced volatile metabolites was evaluated by measuring the inhibition percentages of GVC and AQS of each tested bacterial strain. The experiment was carried out twice with three replicates.

SPME-GC/MS of VOCs
The fresh culture (96 h) of the selected Beauveria isolate was inoculated in a glass tube of 10 mL PDA nutrient media and incubated at 22 • C for 5 days in darkness to collect the volatile organic compounds (VOCs) [69]. The eventually produced VOCs were qualitatively analyzed using the Solid Phase Micro Extraction method (SPME), as discussed below.
The SPME fiber coated with 100 µm of non-grafted poly (dimethylsiloxane) phase (Supelco 57300-U, mounted on a Supelco 57330 support; Merck KGaA, Darmstadt, Germany), was conditioned for 1 h at 250 • C in a stream of helium. A blank run was performed after each analysis to confirm that no residual compounds were polluting the fiber or the column. The fiber was later introduced to the injection port of an HP6890 plus gas chromatograph equipped with a Phenomenex Zebron ZB-5 MS capillary column (30 m × 0.25 mm ID × 0.25 µm film thickness). An HP 5973 mass-selective (mass range: 15-800 mAU; scan rate: 1.9 scan/s; EM voltage: 1435) was used as a detector, whereas helium at 0.8 mL/min was used as a carrier gas. The injection port, equipped with a glass insert (internal diameter 0.75 mm), was split at 250 • C. A desorption time of 1.0 min was used. The detector was maintained at 230 • C. Oven was maintained at 80 • C for 3 min, then the temperature was increased to 250 • C (20 • C/min) for 10 min. All the analyses were performed in triplicate. The chromatograms obtained from the total ion current were integrated without any correction for coelutions, and the results were expressed as a percent of the total area of peaks [70]. All peaks were identified from their mass spectra by comparison with those present in Wiley 275 and NIST 02 libraries [28]. PDA media (not inoculated with the fungus) was used as a negative control. The analysis was carried out twice with three replicates (different injections).

Statistical Analysis
The obtained results for the biological assays were statistically analyzed, applying one-way ANOVA using the Package for the Social Sciences (SPSS) version 13.0 (Prentice Hall: Chicago, IL, USA, 2004). Tukey B Post-Hoc multiple comparison test was used to evaluate the significance level with a probability of p < 0.05.

Conclusions
B. bassiana, apart from being a notable entomopathogenic fungi or biocontrol agent against some phytopathogens, by itself or through its bioactive metabolites. In particular, B. bassiana or its bioactive metabolites could also be used efficiently to control several bacteria in the agronomic field, where the use of antibiotics is forbidden, especially in organic farming. In addition, B. bassiana could also be a useful biocontrol agent against MDR microorganisms to different antibiotics, which are considered a dominant medical problem worldwide. The obtained results from the current research concluded that B. bassiana UniB2439-3 was able to produce some interesting VOCs, such as β-elemene, which has been reported previously to have a strong antimicrobial effect against several pathogenic microorganisms. The ability of B. bassiana to produce the above-mentioned metabolites can underline its antagonistic activity against several phytopathogens, as reported previously in the bibliographic research. Future studies remain necessary to evaluate the in vivo antimicrobial activity of each identified bioactive VOC from B. bassiana against serious phytopathogens, considering that the use of antibiotics is forbidden in agriculture in many countries. Therefore, the search for possible natural alternatives as efficient antimicrobial agents remains necessary.